We have identified and characterized two B. pertussis proteins, tracheal colonization factor (Tcf) and Vag8. Recent work has shown that these proteins and two other B. pertussis bvg-regulated proteins BrkA and pertactin, belong to a family of proteins which share structural homology at their C termini. Each member of this family contains an RGD motif, and the C-terminal 30-Da of these proteins are homologous. The mature, proline rich, 60 kDa form of Tcf is derived from a precursor molecule, which migrates with apparent molecular weights of 92 and 90 K, after cleavage of a C-terminal 30 kDa form. The C-terminal portion of Tcf can be detected as a major protein in outer membrane enriched preparations of B. pertussis strains. The amino acid sequence of this 30 kDa fragment has 58% identity to that of pertactin. Tcf appears to have a role in tracheal colonization, since using a mouse aerosol model of pertussis an 18323 derivative, defective in Tcf, SK34, did not colonize the tracheas as well as the parent. We are currently investigating the role of the RGD motif in tracheal colonization. DNA sequences homologous to tcfA have only been found in strains of B. pertussis. Polyclonal antisera raised to the unique N-terminal portion of Tcf does not recognize any protein in whole-cell lysates of B. bronchiseptica or B. parapertussis. Thus, Tcf appears to be a unique virulence factor of B. pertussis. Bordetella pertussis expresses a bvg-regulated 95 kDa protein, Vag8, encoded by vag-8. B. pertussis SK8 is a TnphoA insertion mutant which no longer expresses Vag8. We have found that SK8 contains two copies of 'phoA, which were distinguished using pSKCAT to produce regulated chloramphenicol acetyl transferase activity. The derived amino acid sequence of vag-8 predicts a 94.8 kDa protein. The C-terminal 30 kDa of this protein shows sequence similarity to the C-terminal sequences of pertactin, Tcf and BrkA which are cleaved from the mature protein. Antisera raised to a fusion of maltose binding protein to the N-terminal 60 kDa of Vag8 recognizes the 95 kDa protein in immunoblots of B. pertussis and B. bronchiseptica. Southern blot analysis of chromosomal DNA from strains of B. pertussis, B. bronchiseptica and B. parapertussis using a probe derived from vag-8 showed hybridization to DNA from all the species. A derivative of B. pertussis 18323 containing an internal deletion and insertion of a Knr cassette in vag-8 was constructed by allelic exchange. When this strain was analysed in the mouse aerosol model of pertussis this 95 kDa negative strain colonized mice as efficiently as the parent B. pertussis strain. edAcute and convalescent sera from unvaccinated children diagnosed with pertussis have been evaluated to determine whether they have antibodies to these proteins. The convalescent sera showed reactivity with the Tcf protein, however, no reactivity was detected to Vag8. Thus, Tcf is produced during human infection and an antibody response is produced to the protein. This finding may have implications for the development of diagnostic assays for B. pertussis disease based on antigens not present in any of the acellular vaccines.